This application is a competitive renewal for "HIV-1, monocytes and the blood-brain barrier," R01NS36126-01. The major goals of the previous project were to: 1) assay virus-endothelial cell infection and its relationship to blood-brain barrier (BBB) function; 2) uncover putative glial neurotoxins (for example, stromal cell derived factor-one- nitric oxide, platelet activating factor, tumor necrosis factor alpha, among others) that influence monocyte migration into the brain; and 3) by utilizing a blood brain barrier (BBB) and severe combined immune deficiency disease (SCID) mouse model of HIV encephalitis (HIVE) to assess chemokine-mediated monocyte recruitment into the central nervous system. These goals were met. Our current focus is in elucidating the biophysical properties of blood-borne macrophages that regulate leukocyte entry into diseased brain during HIV- associated dementia (HAD). These events are pivotal to viral neuropathogenesis. Clearly, once inside the brain, mononuclear phagocytes (MP) (microglia, parenchymal and perivascular macrophages) serve as the Principal cellular reservoir for HIV. Moreover, following immune activation, MP secrete scores of immune "neurotoxic" factors that damage the BBB and neurophil. If macrophage chemotaxis and/or its transendothelial migration could be halted disease could be abrogated. Until now, little attention was paid to the spatial parameters of cell biophysiology, migration and the mechanisms underlying changes in cell shape and volume. Indeed, we have now demonstrated that HIV-1 infected MDM migrate faster than uninfected cells through organotypic brain cultures and Boyden chamber chemotaxis assays. Both infected and uninfected macrophages express ion channels. Thus, the focus of the current work is to decipher how ion channels effect macrophage cell volume and cytosolic calcium that effect the critical components of MP transendothelial migration. The processes that effect MP trafficking will be explored, in the context of macrophage differentiation, activation and viral infection. In particular, we will ascertain the exact ionic currents (independently and together), which are sensitive to cell volume, shape and movement. These may be measured, in part, by whole-cell and single-channel patch-clamp electrophysiological recording assays. Migration of MP will be performed on replicate cell suspensions through the use of artificial barriers (for example Boyden chemotaxis microchamber assays or a BBB model). Ion channel blockers will assess ways to halt the process of MP migration. To correlate these findings to what could occur in an infected human host, MP migration/invasion will be investigated in organotypic cultures of brain slices and in a SCID mouse model of HIVE. DiIC18 stained human monocytes (red) will be co-cultivated with DiOC18-stained organotypic brain slices (green). Cell migration will be evaluated by scoring DiIC18-stained monocytes under laser confocal microscopy (through serial optic sectioning of human or mouse brain tissue). The influence of cell activation and viral infection for the migratory process will also be assessed. Overall, we propose that MP can adjust their cell shape and volume to facilitate their invasion into narrow extracellular spaces of the brain. We hypothesize that these changes require alterations in ion fluxes resulting in water loss and cell shrinkage. Such studies are novel and to our knowledge have not been previously proposed for HAD. The data acquired could provide new insights and therapeutic intervention strategies for monocyte BBB trafficking during progressive HIV-1 infection in brain tissue.